Nanoparticle-sensitized photoporation is an upcoming approach for intracellular delivery of biologics, combining high efficiency and throughput with excellent cell viability. However, as it relies on close contact between nanoparticles and cells, its translation towards clinical applications is hampered by safety and regulatory concerns. Here, we show that light-sensitive iron oxide nanoparticles (IONPs) embedded in biocompatible electrospun nanofibers induce membrane permeabilization by photothermal effects without direct cellular contact with IONPs. The photothermal nanofibers are successfully used to deliver effector molecules, including CRISPR/Cas9 ribonucleoprotein complexes and siRNA, in adherent and suspension cells, including embryonic stem cells and hard-to-transfect T-cells without affecting cell proliferation or phenotype. In vivo experiments furthermore demonstrate successful tumor regression in mice treated with CAR-T cells in which expression of PD1 is downregulated after nanofiber photoporation with siPD1. In conclusion, cell membrane permeabilization with photothermal nanofibers is a promising concept towards the safe and more efficient production of engineered cells for therapeutic applications, including stem cell or adoptive T cell therapy.

Efficient and safe cell engineering by transfection of nucleic acids remains one of the long-standing hurdles for fundamental biomedical research and many new therapeutic applications, such as CAR T cell-based therapies. mRNA has recently gained increasing attention as a more safe and versatile alternative tool over viral- or DNA transposon-based approaches for the generation of adoptive T cells. However, limitations associated with existing nonviral mRNA delivery approaches hamper progress on genetic engineering of these hard-to-transfect immune cells. In this study, we demonstrate that gold nanoparticle-mediated vapor nanobubble (VNB) photoporation is a promising upcoming physical transfection method capable of delivering mRNA in both adherent and suspension cells. Initial transfection experiments on HeLa cells showed the importance of transfection buffer and cargo concentration, while the technology was furthermore shown to be effective for mRNA delivery in Jurkat T cells with transfection efficiencies up to 45%. Importantly, compared to electroporation, which is the reference technology for nonviral transfection of T cells, a fivefold increase in the number of transfected viable Jurkat T cells was observed. Altogether, our results point toward the use of VNB photoporation as a more gentle and efficient technology for intracellular mRNA delivery in adherent and suspension cells, with promising potential for the future engineering of cells in therapeutic and fundamental research applications.

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The modification of CD4+ T cells with exogenous nucleic acids or proteins is a critical step in several research and therapeutic applications, such as HIV studies and cancer immunotherapies. However, efficient cell transfections are not always easily achieved when working with these primary hard-to-transfect cells. While the modification of T cells is typically performed by viral transduction or electroporation, their use is associated with safety issues or cytotoxicity. Vapor nanobubble (VNB) photoporation with sensitizing gold nanoparticles (AuNPs) has recently emerged as a new technology for safe and flexible cell transfections. In this work, we evaluated the potential of VNB photoporation as a novel technique for the intracellular delivery of macromolecules in primary human CD4+ T cells using fluorescent dextrans as model molecules. Our results show that VNB photoporation enables efficient delivery of fluorescent dextrans of 10 kDa in Jurkat (>60% FD10+ cells) as well as in primary human CD4+ T cells (±40% FD10+ cells), with limited cell toxicity (>70% cell viability). We also demonstrated that the technique allows the delivery of dextrans that are up to 500 kDa in Jurkat cells, suggesting its applicability for the delivery of biological macromolecules with a wide range of molecular weights. Altogether, VNB photoporation represents a promising technique for the universal delivery of macromolecules in view of engineering CD4+ T cells for use in a wide variety of research and therapeutic applications.

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Safe and efficient production of chimeric antigen receptor (CAR)‐T cells is of crucial importance for cell‐based cancer immunotherapy. Physical transfection methods have quickly gained in importance in the context of transfecting T‐cells, since they are readily compatible with different cell types and a broad variety of cargo molecules. In particular, nanoparticle‐sensitized photoporation has been introduced in recent years as a gentle yet efficient method to transiently permeabilize cells, allowing subsequent entry of external cargo molecules into the cells. Gold nanoparticles (AuNPs) have been used the most as photothermal sensitizers because they can easily form laser‐induced vapor nanobubbles, a photothermal phenomenon that is shown to be particularly efficient for permeabilizing cells. However, as AuNPs are not biodegradable, clinical translation is hampered. Here, for the first time, the possibility to form laser‐induced vapor nanobubbles from biocompatible polymeric nanoparticles is reported. Compared to electroporation, the most used physical transfection method for T cells, 2.5 times more living mRNA transfected human T cells are obtained via photoporation sensitized by polydopamine nanoparticles. This shows that photoporation is a viable approach for efficiently producing therapeutic engineered T‐cells at a throughput easily exceeding 105 cells per second.

Modern molecular medicine demands techniques to efficiently deliver molecules directly into mammalian cells. As proteins are the final mediators of most cellular pathways, efficient intracellular protein delivery techniques are highly desired. In this respect, photoporation is a promising recent technique for the delivery of proteins directly into living cells. Here, we show the possibility to deliver a model saccharide (FD70) and a model protein (FITC-BSA) into murine B16 melanoma cells by using the vapor nanobubble photoporation technique with an efficiency of 62% and 38%, respectively. Next, we delivered the mixed-lineage kinase domain-like (MLKL) protein, the most terminal mediator of necroptosis currently known, and caspase-8 and -3 protein, which are important proteins in the initiation and execution of apoptosis. A significant drop in cell viability with 62%, 71% and 64% cell survival for MLKL, caspase-8 and caspase-3, respectively, was observed. Remarkably, maximal cell death induction was already observed within 1 h after protein delivery. Transduction of purified recombinant MLKL by photoporation resulted in rapid cell death characterized by cell swelling and cell membrane rupture, both hallmarks of necroptosis. As necroptosis has been identified as a type of cell death with immunogenic properties, this is of interest to anti-cancer immunotherapy. On the other hand, transduction of purified recombinant active caspase-3 or -8 into the tumor cells resulted in rapid cell death preceded by membrane blebbing, which is typical for apoptosis. Our results suggest that the type of cell death of tumor cells can be controlled by direct transduction of effector proteins that are involved in the executioner phase of apoptosis or necroptosis.

Fluorescence-based quantification of messenger RNA and plasmid DNA decay kinetics in extracellular biological fluids and cell extracts